This project is aimed at understanding the molecular basis of intracellular iron metabolism. The cis and trans elements mediating the iron-dependent alterations in abundance of ferritin and the transferrin receptor have been identified and characterized in previous years in this laboratory. Iron-responsive elements (IREs) are RNA stem-loops found in the 5' end of ferritin mRNA and the 3' end of transferrin receptor mRNA. We have cloned, expressed, and characterized an essential iron-sensing protein, the iron-responsive element binding protein (IRE-BP). The IRE-BP binds IRE's when iron levels are depleted, resulting in the inhibition of translation of ferritin mRNA and prolongation of the half-life of the transferrin receptor mRNA. The IRE-BP is 30% identical in amino acid sequence to aconitase, a mitochondrial Krebs cycle enzyme. Mitochondrial aconitase has previously been purified and crystallized and all active site residues are identical in the two proteins. Three of the active site residues are cysteines that ligate an iron-sulfur cluster that has the relatively unusual feature of containing a labile fourth iron. The IRE-BP has aconitase activity, and in vitro manipulations of iron result in changes in RNA binding. In vivo, reciprocal regulation of aconitase activity and RNA binding can be seen when the IRE-BP is over-expressed in a stable cell line. Regulation of RNA binding activity involves a transition from a [4Fe-4S] that does not bind RNA and is an active aconitase to a form that loses iron and aconitase activity. Controlled degradation of the iron-sulfur cluster reveals that the apoprotein is the physiologically relevant form of the protein in iron-depleted cells. Thus, the cluster appears to be undergoing constant disassembly, and reassembly depends on the presence of sufficient iron pools. The main objective of this project is to completely understand the mechanism by which changes in levels of intracellular iron result in changes in RNA binding of the IRE-BP. A variety of techniques will be employed to characterize the iron-sulfur cluster and the RNA binding site of the protein. Over-expression systems will be developed and we will attempt to crystallize the IRE-BP and co-crystallize the protein with the IRE. In addition, we have confirmed that the role of a cDNA cloned at the same time as the initial IRE-BP encodes a second IRE-BP which is rapidly degraded in cells that are iron replete. We are determining the physiologic role of these two proteins through creation of cell lines that stably express the IRP. In addition, "knockouts" of these genes are being created in mice. We intend to use the "knockout" mice to fully characterize the physiology of iron regulation.